Application of anacycline A in preparation of analgesic drugs

Abstract

The invention relates to application of anacycline A in preparation of analgesic drugs, in particular to an anacycline A compound separated from anacycline roots. The compound can be applied to analgesic drugs with inhibition effects on transient receptor potential cation channel subfamily M member 8 (TRPM8), a voltage-gated potassium ion channel 1.2 (Kv1.2), a voltage-gated potassium ion channel 1.3 (Kv1.3), transient receptor potential cation channel protein 6 (TRPC6) and a voltage-gated calcium ion channel 2.1 (Cav2.1), in particular to drugs for neuropathic pain, traumatic pain or inflammatory pain. The compound anacycline A can be combined with a plurality of analgesia-related targets, so that a synergistic effect is generated, the treatment effect is enhanced, and meanwhile, side effects and drug resistance are reduced.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of anazithromycin A in the preparation of analgesic drugs. Background Technology

[0002] It is estimated that approximately 600 million people worldwide suffer from neuropathic pain and cancer-related pain. Currently, opioids remain indispensable in the treatment of these conditions, especially pain from advanced cancer. However, their addictive potential, respiratory depression, cold hyperalgesia, and public health hazards urgently require attention. Therefore, the development of non-addictive, high-efficiency non-opioid analgesics is crucial. Currently, three main strategies have been reported for the development of non-opioid analgesics: developing spinal cord inhibitory synaptic enhancers such as γ-aminobutyric acid (GABA) modulators; implementing neuroimmune axis blockades such as CC chemokine receptor 2 (CCR2) and CC chemokine receptor 5 (CCR5) antagonists; and exploring novel peripheral analgesic targets such as TRPM8 and voltage-gated sodium ion channel 1.8 (Nav1.8).

[0003] TRPM8 is an ion channel mediating cold sensation and pain perception, distributed in sensory neurons in the dorsal root ganglion, trigeminal ganglion, and prostate. Under physiological conditions, low temperatures below 28°C or menthol activation of TRPM8 triggers calcium ion influx, mediating cold sensation transmission. However, under pathological conditions such as nerve damage and chemotherapy-induced neuropathy, excessive activation of TRPM8 leads to cold hyperalgesia, causing severe pain even with mild cold stimuli. TRPM8 antagonists selectively block the channel, inhibit calcium influx, directly suppress abnormal neuronal firing, and downregulate central sensitization mediated by the spinal cord metabolite glutamate receptor (mGluR) pathway, reversing cold hyperalgesia and thus blocking neurogenic, cancerous, and inflammatory pain signals. However, existing antagonists (such as VBJ103) have limited clinical research due to potential hypothermic side effects and low bioavailability. Notably, the Kv1.2 potassium channel plays a crucial role in the transmission of action potentials triggered by cold stimuli by regulating the cold sensation threshold through molecular interaction with TRPM8. Simultaneous targeting of TRPM8 and Kv1.2 synergistically inhibits calcium influx and potassium efflux, significantly reducing neuronal excitability and peripheral nociceptive signal transduction. This strategy not only effectively inhibits pain transmission and provides non-opioid analgesia, but also avoids adverse reactions such as hypothermia caused by inhibiting TRPM8 alone.

[0004] Therefore, developing novel non-opioid multi-target potent analgesics that simultaneously act on TRPM8 and Kv1.2 is of great value. Exploring lead compounds with significant pharmacodynamic activity from traditional medicinal plants is a hot topic in drug research; therefore, discovering novel ion channel inhibitors and analgesic compounds from medicinal plants is an effective approach to developing novel analgesics.

[0005] Anachigenine A is a novel enantiomer of a pair of highly conjugated diamino 6 / 6 / 6 / 6 / 5 pentacyclic alkaloids with a unique 6,13-diazapentacyclic [10.3.3]. 1,3 .1.0 2,7 .0 8,12 The compound has a nonadecane ring skeletal structure, and studies have shown that it possesses certain anti-inflammatory effects. Patent No.: ZL 2023 1 1104612.0 Compounds with anti-inflammatory effects from anachine root, their preparation, and applications. Further research into the bioactivity of this compound revealed good analgesic activity, exerting its analgesic effect by inhibiting multiple analgesic-related targets, including TRPM8, Kv1.2, Kv1.3, TRPC6, and Cav2.1. The compound, anachine base A, is used to inhibit TRPM8, Kv1.2, Kv1.3, TRPC6, and Cav2.1 ion channels. Summary of the Invention

[0006] The purpose of this invention is to provide the application of anachymidine A in the preparation of analgesic drugs. Anachymidine A is a compound isolated from anachymidine root. This compound exhibits inhibitory activity against transient receptor potential cation channel subfamily M member 8 (TRPM8), voltage-gated potassium channel 1.2 (Kv1.2), voltage-gated potassium channel 1.3 (Kv1.3), transient receptor potential cation channel protein 6 (TRPC6), and voltage-gated calcium channel 2.1 (Cav2.1). Specifically, it is used as an analgesic for neuropathic pain, traumatic pain, or inflammatory pain. The compound anachymidine A of this invention can bind to multiple analgesic-related targets, thereby producing a synergistic effect, enhancing therapeutic efficacy, and reducing side effects and drug resistance, thus solving the technical problems of low safety and significant adverse reactions in existing analgesic drugs.

[0007] The present invention relates to the application of anazithromycin A in the preparation of analgesic drugs.

[0008] The application of anazithromycin A in the preparation of analgesic drugs described in this invention is a further study based on the patent number: ZL 202311104612.0, which describes compounds with anti-inflammatory effects in anazithromycin and their preparation and application. The structural formula of anazithromycin A is: dextrorotatory formula (+)-1, levorotatory formula (-)-1, or a mixture of racemic formula (+)-1 and formula (-)-1.

[0009]

[0010] The anarchine A compound (dextral formula (+)-1, levorotatory formula (-)-1, or racemic formula (+)-1 and formula (-)-1) is used in the preparation of analgesic drugs.

[0011] The anachygenin A compound is used to inhibit the use of receptor potential cation channel subfamily M member 8 (TRPM8), voltage-gated potassium channel 1.2 (Kv1.2), voltage-gated potassium channel 1.3 (Kv1.3), transient receptor potential cation channel protein 6 (TRPC6), and voltage-gated calcium channel 2.1 (Cav2.1).

[0012] Compared with existing technologies, the application of anacrine A in the preparation of analgesics according to this invention has the following main technical advantages:

[0013] (1) Regarding analgesic activity, the anazithromycin A compounds (±)-1, (+)-1, and (-)-1 provided in this invention exhibit significant analgesic activity against acetic acid-induced writhing in mice at a dose of 5 mg / kg. Furthermore, at lower doses such as 1 mg / kg, 0.2 mg / kg, and 0.02 mg / kg, the analgesic activity of (±)-1 and (-)-1 is stronger than that of the positive control drug morphine (a first-line analgesic used clinically), suggesting their potential as novel analgesic components or lead compounds with broad development prospects.

[0014] (2) This invention demonstrates through patch-clamp experiments that anarchine A compounds (+)-1 and (-)-1 exert their analgesic activity by inhibiting multiple analgesic targets such as TRPM8, Kv1.2, Kv1.3, TRPC6 and Cav2.1 ion channels. The interaction sites between anarchine A compounds (+)-1 and (-)-1 and TRPM8 and Kv1.2 were analyzed using computer molecular docking technology, providing a theoretical basis for further design and development of novel multi-target analgesics acting on TRPM8 and Kv1.2 ion channels.

[0015] The anarchine A described in this invention has applications in analgesic drugs, specifically involving analgesic activity experiments, target verification, and molecular docking of anarchine A (formula (+)-1, formula (-)-1).

[0016] The present invention provides anachye root base A having the following forms: dextrorotatory formula (+)-1, levorotatory formula (-)-1, or a mixture of racemic formula (+)-1 and formula (-)-1;

[0017]

[0018] Compound (±)-1: N-(1-(2,2,5,5,8,12,12-heptamethyl-3,7-dione-1,2,3,4,5,6,7,8,9,11,12-decahydro-6H,13H-9a,14a-methyl-bridged cyclopentano[7,8]aza[4,5,6-ij]isoquinoline-2-yl)-2-methylpropyl-2-yl)acetamide;

[0019] Compound (+)-1: N-(1-((8R,9aS,10aS)-2,2,5,5,8,12,12-heptamethyl-3,7-dione-1,2,3,4,5,6,7,8,9,11,12-decahydro-6H,13H-9a,14a-methyl-bridged cyclopentano[7,8]aza[4,5,6-ij]isoquinoline-2-yl)-2-methylpropyl-2-yl)acetamide;

[0020] Compound (–)-1 is: N-(1-((8S,9aR,10aR)-2,2,5,5,8,12,12-heptamethyl-3,7-dione-1,2,3,4,5,6,7,8,9,11,12-decahydro-6H,13H-9a,14a-methylbridgedcyclopentano[7,8]aza[4,5,6-ij]isoquinoline-2-yl)-2-methylpropyl-2-yl)acetamide.

[0021] This invention provides experimental data on the analgesic activity of anachymidine A compounds (±)-1, (+)-1, and (-)-1. The analgesic activity of anachymidine A compounds (±)-1, (+)-1, and (-)-1 was determined by acetic acid-induced writhing test in mice. The results showed that compounds (±)-1, (+)-1, and (-)-1 exhibited significant analgesic activity at a dose of 5 mg / kg; and at lower doses such as 1 mg / kg, 0.2 mg / kg, and 0.02 mg / kg, the analgesic activity of (±)-1 and (-)-1 was stronger than that of the positive control drug morphine (a first-line analgesic used clinically).

[0022] This invention screened potential targets of anachygen base A compound (+)- / (-)-1 and demonstrated through patch-clamp experiments that compound (+)- / (-)-1 is an inhibitor of TRPM8, Kv1.2, Kv1.3, TRPC6 and Cav2.1.

[0023] This invention utilizes molecular docking software such as Autodock Vina and Discovery Studio to study the interaction mode of anachine base A compound (+)- / (-)-1 with TRPM8 (8E4Q) and Kv1.2 (5WIE); and further confirms the binding mechanism of compound (-)-1 with TRPM8 through molecular dynamics simulations. Attached Figure Description

[0024] Figure 1 The figure shows the analgesic activity of anazithromycin A compounds (±)-1, (+)-1, and (-)-1 of the present invention at doses of 5.0, 1.0, 0.2, and 0.04 mg / kg against an acetic acid-induced mouse writhing model. In the figure, (A) represents the number of writhing episodes in mice within 30 minutes at different concentrations of the compound, (B) represents the percentage inhibition rate of analgesia at different concentrations of the compound, and NT indicates not tested.

[0025] Figure 2 The figures show the experimental results of the blocking of TRPM8, Kv1.2, Kv1.3, TRPC6, and Cav2.1 ion channels by anachine A(+)- / (-)-1 of this invention. (A) shows the peak current inhibition curves of TRPM8 at different concentrations of (+)-1, and (B) shows the IC50 of the inhibitory effect of (+)-1 on TRPM8. 50 The value curves are shown in (C) and (D) respectively. (C) shows the peak current suppression curves of TRPM8 at different concentrations of (-)-1, and (D) shows the IC50 value of the inhibitory effect of (-)-1 on TRPM8. 50 (E) Suppression curves of different concentrations of (-)-1 on the peak current of Kv1.2; (F) IC50 curves showing the suppression effect of (-)-1 on Kv1.2. 50 Value curve.

[0026] Figure 3 This diagram shows the molecular docking results of anachygen base A compound (+)- / (-)-1 with TRPM8. In the diagram, (A) is a schematic diagram of compound (+)-1 embedding into the TRPM8 channel entrance pore via electrostatic surface interaction; (B) is a three-dimensional structural schematic diagram of the interaction between compound (+)-1 and key residues of the TRPM8 channel; (C) is a two-dimensional structural schematic diagram of the interaction between (+)-1 and key residues of the TRPM8 channel; (D) is a schematic diagram of (-)-1 embedding into the TRPM8 channel entrance pore via electrostatic surface interaction; (E) is a three-dimensional structural schematic diagram of the interaction between (-)-1 and key residues of the TRPM8 channel; and (F) is a three-dimensional structural schematic diagram of the interaction between (-)-1 and key residues of the TRPM8 channel. The green, light green, and purple circles and lines represent hydrogen bonding, van der Waals forces, and π-alkyl interactions, respectively.

[0027] Figure 4This diagram shows the molecular docking results of anachygen base A compound (+)- / (-)-1 with Kv1.2. In the diagram, (A) is a schematic diagram of compound (+)-1 embedding into the entrance pore of the Kv1.2 channel via electrostatic surface interaction; (B) is a three-dimensional structural schematic diagram of the interaction between compound (+)-1 and key residues of the Kv1.2 channel; (C) is a two-dimensional structural schematic diagram of the interaction between (+)-1 and key residues of the Kv1.2 channel; (D) is a schematic diagram of (-)-1 embedding into the entrance pore of the Kv1.2 channel via electrostatic surface interaction; (E) is a three-dimensional structural schematic diagram of the interaction between (-)-1 and key residues of the Kv1.2 channel; and (F) is a three-dimensional structural schematic diagram of the interaction between (-)-1 and key residues of the Kv1.2 channel. The green, light green, and purple circles and lines represent hydrogen bonds, van der Waals forces, and π-alkyl interactions, respectively. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0029] Example 1

[0030] The analgesic activity of anachymosis base A compounds (±)-1, (+)-1, and (-)-1 was evaluated using an acetic acid-induced mouse writhing model.

[0031] Half male and half female Kunming mice were randomly divided into three groups (n=10 / group). The control group was injected intraperitoneally with 0.1 ml / 10 g of physiological saline, the morphine group was injected intraperitoneally with 5, 1, 0.2 or 0.04 mg / kg of morphine, and the test compound group was injected intraperitoneally with 5, 1, 0.2 or 0.04 mg / kg of the compound in physiological saline solution. Thirty minutes after administration, each group of mice was injected intraperitoneally (ip) with 0.8% (v / v) acetic acid solution (0.1 ml / 10 g), and the cumulative number of writhing responses within 30 minutes was immediately recorded. The writhing inhibition rate was calculated using the formula: Writhing inhibition rate (%) = [(number of writhing responses in the control group - number of writhing responses in the drug group) / number of writhing responses in the control group] × 100%.

[0032] Conclusion: The analgesic activity of anachine A compounds (±)-1, (+)-1, and (-)-1 was evaluated as follows: Figure 1As shown; all tested anachine A compounds exhibited significant analgesic activity at doses of mg / kg, with writhing inhibition rates ranging from 54.8% to 96.6%; at a dose of 1 mg / kg, the inhibition rates of (±)-1 (74.9% ± 0.8%), (-)-1 (88.1% ± 0.8%), and (+)-1 (89.9% ± 1.7%) were significantly higher than those of morphine (73.8% ± 1.3%); notably, at a clinically equivalent dose of 0.2 mg / kg (the therapeutic dose of morphine), (±)-1 (59.9%) showed significantly higher inhibition rates. The analgesic activity of (±)-1 (8%±0.8%) and (-)-1 (74.9%±0.8%) was significantly better than that of morphine (56.5%±2.3%); even at the lowest test dose of 0.04 mg / kg, (±)-1 (51.6%±1.8%) and (-)-1 (57.7%±1.1%) still maintained a higher inhibition rate than morphine (44.2%±2.5%); chiral selectivity analysis showed that the analgesic activity of the levorotatory isomer (-)-1 was significantly stronger than that of the dextrorotatory isomer (+)-1 and the racemic isomer (±)-1 at all doses.

[0033] Example 2

[0034] The inhibitory activities of anachine A compound (+)- / (-)-1 on TRPM8, Kv1.2, Kv1.3, TRPC6, and Cav2.1 were evaluated using patch-clamp electrophysiological experiments.

[0035] HEK293T cells (ATCC cell bank) were used for experiments. HEK293T cells were cultured at 37°C in an incubator with 5% carbon dioxide in a DMEM / Ham's F-12 basal medium supplemented with 10% fetal bovine serum. For electrophysiological assays, experimental data were acquired using an EPC-10 amplifier (HEKA) and stored in PatchMaster (HEKA) software. Patch-clamp recordings were performed using an inverted microscope (Mshot, China) and an MP225 microelectrode manipulator (Sutter Instruments, USA). Recording electrodes were brought into contact with cells to form a GΩ seal, and then whole-cell recording mode was used. All experiments were performed at room temperature.

[0036] Conclusion: At a concentration of 10 μmol / L, anachine A compound (+)-1 showed significant inhibitory effects on TRPM8, with an inhibition percentage of 68.76% ± 0.50%, moderate inhibitory effects on Kv1.2, with an inhibition percentage of 30.95% ± 1.17%, and weak inhibitory effects on TRPC6, Cav2.1, and Kv1.3, with inhibition rates of 3.77% ± 0.62%, 9.40% ± 6.12%, and 4.50% ± 2.02%, respectively (see Table 2).

[0037] Table 2. Percentage inhibition of five analgesic-related ion channels by anachygenin A compounds (+)-1 and (-)-1. (Two replicates, concentration 10 μmol / L)

[0038]

[0039] In contrast, anachygenin A compound (-)-1 showed significant inhibitory effects on TRPM8 and Kv1.2 channels, with inhibition percentages of 68.37% ± 5.88% and 46.20% ± 1.19%, respectively (see Table 2); moderate inhibitory effects on Cav2.1, with an inhibition rate of 21.20% ± 6.08%; weak inhibitory effects on Kv1.3, with an inhibition rate of 12.26% ± 5.07%; and weak inhibitory effects on TRPC6, with an inhibition rate of 3.94% ± 0.76%. Anachygenin A compounds (+)-1 and (-)-1 inhibited the peak current of TRPM8 in a dose-dependent manner, with their half-maximum inhibitory concentration (IC50) being... 50 The concentrations were 1.90±0.09 and 1.40±0.17 μmol / L, respectively. Figure 3 Anachygenine A compound (-)-1 also blocked the Kv1.2 peak current in a dose-dependent manner, with a half-inhibition concentration (IC50) of 1 / 3. 50 The value was 14.40±0.62 μmol / L, indicating that anachine A compounds (+)-1 and (-)-1 exert their analgesic activity by inhibiting multiple analgesic targets such as TRPM8, Kv1.2, Kv1.3, TRPC6 and Cav2.1 ion channels.

[0040] Example 3

[0041] This embodiment uses Autodock 4.2.6 molecular docking software (The Scripps Research Institute, USA) to study the interaction modes of anachine base A compounds (+)-1 and (-)-1 with TRPM8 (8E4Q) and Kv1.2 (5WIE). Through molecular dynamics simulation, the dynamic conformational changes and trajectory of compound (-)-1 binding with TRPM8 protein are reflected.

[0042] Conclusion: Molecular docking results showed that anachine base A compounds (+)-1 and (-)-1 occupied almost identical positions within the TRPM8 pocket and exhibited almost identical binding modes; both were bound to phenylalanine 846 (Phe846), isoleucine 746 (Ile746), leucine 842 (Leu842), tyrosine 835 (Tyr835), threonine 839 (Thr839), alanine 879 (Ala879), and valine 878 (…). Residues such as Val878 exhibit hydrophobic interactions and form π-alkyl interactions with residues such as leucine 749 (Leu749), leucine 750 (Leu750), alanine 874 (Ala874), phenylalanine 87 (Phe87), and valine 875 (Val875); however, there are certain chiral differences among them, the main difference being the orientation of the C-17 carbonyl group of acetamide; the C-17 carbonyl group in anachine base A compound (-)-1 is extended It extends into a hydrophobic region, forming hydrogen bonds with tyrosine 907 (Tyr907) residues and hydrophobically interacting with methionine 877 (Met877) residues; however, anachygenine A compound (+)-1 does not exhibit such interactions; this difference can reasonably explain the stronger inhibitory effect of anachygenine A compound (-)-1 on TRPM8; similar to TRPM8, anachygenine A compounds (+)-1 and (-)-1 interact with Kv1.2... The binding modes of the active pockets are almost identical. They all exhibit hydrophobic interactions with three residues: valine 89 (Val89), tyrosine 90 (Tyr90), tryptophan 121 (Trp121), and tryptophan 243 (Trp243); π-alkyl interactions with two residues: tryptophan 57 (Trp57) and tryptophan 272 (Trp272); and hydrogen bonds with two residues: arginine 189 (Arg189) and lysine 276 (Lys276). The chiral difference lies in the spatial orientation of the C-17 carbonyl group. In (-)-1, the extended conformation of the C-17 carbonyl group forms two hydrogen bonds with the Arg189 residue. In contrast, in the (+)-1 compound of anachygan base A, the C-17 carbonyl group forms only one hydrogen bond with the Arg189 residue. This difference may explain why (-)-1 exhibits a stronger inhibitory effect on the Kv1.2 channel than (+)-1.

[0043] To further confirm the binding mechanism between anachine base A compound (-)-1 and the TRPM8 ion channel, molecular dynamics simulations of the (-)-1 / TRPM8 complex were performed using Discovery Studio software. The results showed that the conformation of anachine base A compound (-)-1 within the TRPM8 binding site was highly stable, with no significant conformational changes observed. The root mean square deviation (RMSD) of the complex remained below 0.25 nm, and the maximum root mean square fluctuation (RMSF) did not exceed 1.2 nm, thus confirming the dynamic stability of the complex structure.

[0044] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of anazithromycin A in the preparation of analgesic drugs.

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